This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The heavy metal cadmium is a widespread environmental contaminant that accumulates in the body and poses a threat to human health, especially its impact on the development of bone diseases, including osteoporosis. There are several sources of cadmium emission into the environment;notable is the global increase in disposal of electronic-waste (e.g., cell phones and computers) containing this toxic metal. Humans are exposed to cadmium through smoking cigarettes and ingesting contaminated food and water. Despite its recognized importance as an environmental toxin, little is known about how cadmium directly impacts bone cells, in particular bone-forming osteoblasts. We previously reported that cadmium induces apoptosis in human osteoblast-like cells, Saos-2, through caspase-3 activation. To clarify the impact of cadmium in bone, the proposed research explores the role of oxidative stress in cadmium-induced apoptosis and investigate cadmium's impact on mineralization using nontumor derived MC3T3-E1 and tumor derived Saos-2 osteoblasts. There is growing evidence that oxidative stress contributes to a number of age-related diseases, including osteoporosis, and that antioxidants function to mitigate the damaging effect of oxidative stress. Our hypothesis is that cadmium exposure results in oxidative stress which leads to reduced osteoblast mineralization and increased apoptosis. We predict that pretreatment with antioxidants will protect against cadmium-induced osteotoxicity. Specific aim 1 is to determine if estradiol has antioxidant properties and protects against cadmium-induced oxidative damage and osteoblast apoptotic death. Specific aim 2 is to determine if Idaho sagebrush-derived flavonoids have antioxidant properties and protect against cadmium-induced oxidative damage and osteoblast apoptotic death. Specific aim 3 will determine whether cadmium exposure leads to reduced osteoblast mineralization and whether estradiol or Idaho sagebrush-derived flavonoids protect against cadmium-induced decrease in mineralization. We will examine the effect of CdCl2 exposure on the expression of key osteoblast genes (e.g., RUNX2, osteopontin, osteocalcin, and alkaline phophastase) by real-time PCR, and specific markers for mineralization including von Kossa and Alizarin red staining. This research will provide insight into mechanisms underlying cadmium-induced osteotoxicity, in particular the role of oxidative stress. These are fundamental studies in the mechanisms of "Cell Signaling" in osteoblast development and apoptosis. Knowing the mechanisms by which cadmium disrupts osteoblast function, and the ability of estradiol and sagebrush-derived flavonoids to act as antioxidants, will provide a better understanding of how environmental toxins contribute to bone disease and the ability of antioxidants to reduce the negative impacts of toxins on bone health. A long term goal is to develop an adult zebrafish model to validate in vitro studies and link cadmium's impact in osteoblasts to net bone loss.